CN110462036A - A kind of novel D-Psicose 3- epimerase and the method for preparing D-Psicose using the enzyme - Google Patents
A kind of novel D-Psicose 3- epimerase and the method for preparing D-Psicose using the enzyme Download PDFInfo
- Publication number
- CN110462036A CN110462036A CN201780077035.7A CN201780077035A CN110462036A CN 110462036 A CN110462036 A CN 110462036A CN 201780077035 A CN201780077035 A CN 201780077035A CN 110462036 A CN110462036 A CN 110462036A
- Authority
- CN
- China
- Prior art keywords
- psicose
- epimerase
- ala
- gly
- microorganism
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y501/00—Racemaces and epimerases (5.1)
- C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The method for providing the new D-Psicose 3- epimerase of one kind and preparing psicose using the enzyme.
Description
Technical field
The method that following discloses are related to D-Psicose 3- epimerase and prepare D-Psicose using it.
Background technique
D-Psicose (hereinafter referred to " psicose ") is a kind of monosaccharide, it is known that it is in nature with very small
Measure existing rare sugar.Its heat is almost nil, while having nearly 70% sugared sugariness, and since its function such as inhibits blood
Sugar and inhibition lipid synthesis etc., have had received widespread attention as new food composition.
Due to these characteristics, psicose, which is taken into account in various food, is used as sweetener replacement sugar.However, due to A Luo
Ketose exists in nature with very small amount, and the demand to the method for effectively preparing psicose increasingly increases.
A kind of method that the known method for preparing psicose is catalyzed including the use of molybdenum acid ion (Bilik, V.,
Tihlarik,K.,1973,Reaction of Saccharides Catalyzed by Molybdate
Ions.IX.Epimerization of Ketohexoses.Chem.Zvesti.28:106-109), it is a kind of by by ethyl alcohol and
Triethylamine heats and is prepared by D-Fructose together chemical method (Doner, L.W., 1979, the Isomerization of psicose
of D-Fructose by Base:Liquid-Chromatographic Evaluation and The Isolation of
D-Psicose.Carbohydr.Res.70:209-216) and it is a kind of using generate D-Psicose 3- epimerase it is micro-
Biology is prepared the biological method (Korean Patent Publication No. 10-2011-0035805) of psicose by D-Fructose.Pass through chemistry
Method prepares the problem of psicose and is, generates a large amount of by-product, and therefore, it is necessary to carry out complicated purifying.In addition, biological
There is also yield is very low and the high problem of preparation cost for method.
In these cases, the present inventor has paid a large amount of effort to develop a kind of preparation yield of raising psicose
Method, and as a result, it was confirmed that when using new D-Psicose -3- epimerase (hereinafter referred to " A Luo ketone of the invention
Sugared epimerase ") when, the rate that D-Fructose is converted into psicose (is hereinafter referred to converted into psicose from D-Fructose
Conversion rate) increase, so as to improve the preparation yield of psicose significantly, and complete the present invention.
Technical problem
One embodiment of the invention is related to providing the new psicose epimerase of one kind, encodes the psicose
The polynucleotides of epimerase, the recombinant vector including the polynucleotides and the microorganism for wherein importing the carrier.
Another embodiment of the invention is related to providing a kind of composition for being used to prepare D-Psicose comprising Ah
Lip river ketose epimerase expresses the microorganism of the psicose epimerase or the culture of the microorganism, and a kind of
The method for preparing D-Psicose using the psicose epimerase.
Technical solution
An exemplary implementation scheme according to the present invention provides a kind of amino acid sequence group by SEQ ID NO:1
At D-Psicose -3- epimerase.
In an exemplary embodiment, which may include having extremely with SEQ ID NO:1
The polypeptide of few 80%, 90%, 95%, 97% or 99% homology.Obviously, there is the activity for converting D-Fructose to psicose
Amino acid sequence with above-mentioned homology may include such situation, wherein the partial amino-acid series quilt of SEQ ID NO:1
Replace, insertion, modify and/or delete.In addition, the polypeptide with psicose epimerase activity also may include and unlimited
In the polypeptide by polynucleotide encoding, all or part of complementary sequence of the polynucleotides and the nucleotide sequence of oded p
Column hybridize under strict conditions, and the probe can be prepared from known sequence, such as psicose difference of the invention is to different
Structure enzyme.
The term as used herein " polynucleotides " refers to polyribonucleotide or polydeoxyribonucleotide, nucleotide list
Body is unmodified or through modifying, and is formed by covalent bond with the nucleotide polymer of long chain extension.
The term as used herein " stringent condition " refers to the condition for allowing specific hybrid between polynucleotides.The condition takes
Certainly in the length of polynucleotides and complementarity.Its parameter be it is known in the art that simultaneously in the literature specifically describe (for example,
J.Sambrook etc., ibid).For example, stringent condition can be enumerated has 80%, 90%, 95%, 97% or 99% or more each other
The condition that the gene of high high homology hybridizes each other, the item that each other there is the gene of the homology lower than it not hybridize each other
Part or the general wash conditions of Southern hybridization, i.e., in salinity and temperature, such as 60 DEG C, 1 × SSC, 0.1%SDS, especially
60 DEG C, 0.1 × SSC, 0.1%SDS, more particularly 68 DEG C, 0.1 × SSC, 0.1%SDS washed once, especially wash two
The secondary condition arrived three times.Probe used in hybridization can be a part of the complementary series of base sequence.Such probe can
Using using the genetic fragment containing base sequence as template, using the oligonucleotides prepared according to known array as primer, by PCR come
Building.In addition, those skilled in the art can according to need the factor of such as probe length, the temperature and salt of washing solution are adjusted
Concentration.
The term as used herein " homology " refers to the two phase same sex percentage between polynucleotides or polypeptide portion.From one
A part can be determined to the homology between the sequence of another part by known technology.For example, homology can pass through
Compare the parameter of the sequence information between two polynucleotide molecules or two peptide molecules directly to determine, such as score, phase
The same sex and similitude etc. are carried out using the computer program (such as BLAST2.0) that sequence information is classified and is easy to get.This
Outside, the hybrid polynucleotide under conditions of homology between polynucleotides can stablize double-strand by being formed between homologous region,
Then it is degraded by single-stranded specific nucleic acid enzyme and is determined with determining the size of degradation fragment.
As long as in addition, protein have corresponding to by formed in the amino acid sequence of SEQ ID NO:1 of the invention Ah
The activity of Lip river ketose epimerase can add nonsense sequence before and after the amino acid sequence of SEQ ID NO:1, or
Including naturally occurring mutation or its silent mutation.Protein including SEQ ID NO:1 is intended to be included within the scope of the present invention.
In addition, D-Psicose -3- epimerase of the invention can by the polynucleotide sequence of SEQ ID NO:2, or
With its have at least 80%, 90%, 95%, 97% or 99% homology polynucleotide sequence coding, but the present invention is not limited to
This.It is further obvious that passing through codon for the polynucleotides for encoding D-Psicose -3- epimerase of the invention
Degeneracy, can translate into the protein being made of SEQ ID NO:1 amino acid sequence or with its protein with homology
Polynucleotides also be included within the scope of polynucleotide sequence of the invention.It will be appreciated by those skilled in the art that known to use
Recombinant technique, volume can be prepared by the nucleotide sequence of substitutions, additions and/or deletions SEQ ID NO:2 one or more
Code has the polynucleotides of of substantially equal active enzyme.
In another exemplary embodiment, psicose epimerase of the invention can belong to derived from Kaistia
Microorganism.Specifically, psicose epimerase of the invention can be derived from Kaistia granuli, more specifically,
Kaistia granuli KCTC12575 can be derived from.
In another exemplary embodiment, psicose epimerase of the invention, which can have, passes through dodecyl
25kDa measured by sodium sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to 37kDa, 27kDa to 35kDa or 30kDa extremely
The molecular weight of 35kDa.
Another exemplary implementation scheme according to the present invention provides coding by the amino acid sequence group of SEQ ID NO:1
At D-Psicose -3- epimerase polynucleotides.
In an exemplary embodiment, polynucleotides provided herein can be by the base sequence of SEQ ID NO:2
Column or the multicore formed with SEQ ID NO:2 with the sequence of 80%, 90%, 95%, 97% or 99% or higher homology
Thuja acid.It is further obvious that, by Codon degeneracy, can be translated by SEQ for polynucleotides provided by the invention
The protein of the amino acid sequence composition of ID NO:1 is also included within this hair with the polynucleotides of its protein with homology
In bright range.
Another exemplary implementation scheme according to the present invention, provides a kind of recombinant vector comprising the coding present invention
D-Psicose 3- epimerase polynucleotides.
Recombinant vector of the invention can have wherein will coding psicose epimerase using known standard method
Polynucleotides be inserted into the form in cloning vector or expression vector.The term as used herein " cloning vector " relates to take
Band DNA fragmentation enters host cell and makes its regenerated carrier.Cloning vector can also include polyadenylation signal, transcribe eventually
Only sequence and/or multiple cloning sites.Here, multiple cloning sites may include at least one in endonuclease and restriction enzyme sites
Kind.In an exemplary embodiment, the polynucleotides for encoding psicose epimerase, which can be located at polyadenylation, to be believed
Number and transcription terminator upstream.The term as used herein " expression vector " is related to cloned DNA in suitable host
DNA sequence dna necessary to transcription and translation.In addition, " expression vector " used herein is related to including operationally connecting with insert
The gene construct of the required controlling element connect, so that the expression when insert is present in subject cell.Term " can operate
Ground connection " refers to, a kind of function, which by another function passes through the polynucleotide sequence on polynucleotides and is associated with, to be adjusted.Use standard
Recombinant DNA technology can prepare and purify expression vector.Expression vector may include promoter, initiation codon, coding born of the same parents it is phonetic
It is at least any in the gene and terminator codon of pyridine epimerase.
Another exemplary implementation scheme according to the present invention, provides a kind of microorganism, wherein having imported as described above
Recombinant vector.
In an exemplary embodiment, import recombinant vector of the invention microorganism can be with include coding Ah
The recombinant vector of the polynucleotides of Lip river ketose epimerase or with including the multicore being made of the base sequence of SEQ ID NO:2
The microorganism of the recombinant vector conversion of thuja acid, the psicose epimerase includes the amino acid sequence of SEQ ID NO:1.
The term as used herein " conversion ", which refers to, imports host cell for gene or recombinant vector including the gene, so that
The gene can express in host cell.The present invention includes the gene of any conversion, as long as the gene of the conversion can be in place
It expresses in chief cell, in the chromosome without limiting its Insertion Into Host Cell or is located at except the chromosome of host cell.This
The method for transformation of invention includes instantaneous conversion, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated turn
Transfection, electroporation, Electricinjection, the chemical treatment etc. that dye, deae dextran mediate, however, the present invention is not limited thereto.Recombination can be used
The host cell of carrier conversion may include prokaryotic cell, plant cell, zooblast etc..It can be used and imported with high DNA
The host cell of the high expression rate of efficiency and the DNA of importing.For example, host cell can be Escherichia coli (E.coli), gemma
Bacillus genus strain, Corynebacterium sp. strain, Salmonella strains etc., for example, can be Escherichia coli such as W3110, BL21,
JM109, K-12, LE392, RR1 and DH5 α etc..More specifically, microorganism of the invention can be with KCCM11918P preservation
E. coli bl21 (DE3)/KGDPE.
Another exemplary implementation scheme according to the present invention provides a kind of composition for being used to prepare D-Psicose,
Comprising: D-Psicose 3- epimerase, the expression D-Psicose 3- epimerase including SEQ ID NO:1
Microorganism, the microorganism culture.
In an exemplary embodiment, microorganism of the invention can be bacterial strain itself, its culture or microorganism
Lysate.Culture or lysate of the invention may include D-Psicose -3- epimerase of the invention.In addition, this
The culture of microorganism of invention may include or not include the microorganism.In addition, the lysate of microorganism of the invention can be with
It is the lysate obtained by cracking microorganism or its culture, or the supernatant obtained by centrifugation lysate.
In another exemplary embodiment, the composition for being used to prepare D-Psicose of the invention can be wrapped further
Include substrate of the D-Fructose as psicose epimerase.
In another exemplary embodiment, microorganism of the invention can be fixed on carrier ready for use.Energy
The example for being enough in carrier of the invention includes, but are not limited to agar, agarose, kappa carrageenan, alginates or deacetylated
Chitin.
In addition, the composition for being used to prepare D-Psicose of the invention, which may further include, can support psicose
Any component of preparation.Specifically, the composition for being used to prepare D-Psicose of the invention may further include metal.More
Specifically, metal of the invention can be at least one metal selected from manganese, calcium, magnesium, iron, lithium and sodium.In addition, of the invention
Metal can be metal ion or metal salt.The concentration of metal of the invention can be 0.1mM~10mM, 0.1mM~7mM,
0.1mM~4mM, 0.5mM~10mM, 0.5mM~7mM, 0.5mM~4mM, 1mM~10mM, 1mM~7mM, 1mM~4mM, 2mM
~10mM, 2mM~7mM, 2mM~4mM.More specifically, metal salt of the invention can be selected from LiCl, Na2SO4、MgCl2、
NaCl、FeSO4、MgSO4、MnCl2、MnSO4And CaCl2At least one metal salt.
Another exemplary implementation scheme according to the present invention provides a kind of method for preparing D-Psicose, comprising: make
The D-Psicose 3- epimerase being made of the amino acid sequence of SEQ ID NO:1, it is poor to express the D-Psicose 3-
It is contacted to the culture of the microorganism of isomerase or the microorganism with D-Fructose.
In an exemplary embodiment, the preparation method can further comprise before being contacted with D-Fructose, it
Metal is added afterwards or simultaneously.
In another exemplary embodiment, which may further include, and is contacting or is adding with D-Fructose
After adding the metal, the product of contact of separation and/or purifying including psicose.Separation and/or purifying can by a kind of or
A variety of known methods carry out, such as dialysis, precipitating, absorption, electrophoresis, ion-exchange chromatography and fractional crystallization etc., but not limited to this.
In addition, preparation method of the invention may further include, before or after separation and/or purifying, taken off
Color and/or desalination.By carrying out decoloration and/or desalination, the free from admixture psicose more refined can be obtained.
In another exemplary embodiment, preparation method of the invention may further include, and connect with D-Fructose
Touching, adds the metal, separation and/or purifying, or after carrying out decoloration and/or desalination, crystallizes to D-Psicose.Knot
Crystalline substance can be carried out by using conventional use of method for crystallising.For example, can be crystallized by using cooling crystallization method.
In another exemplary embodiment, preparation method of the invention may further include, before crystallization, dense
Contracting psicose.Crystalline rate can be improved in the concentration.
In another embodiment, preparation method of the invention can further comprise, after separation and/or purifying,
It contacts unreacted D-Fructose with psicose epimerase, or can further comprise, after crystallization, reuse mother
Liquid separates crystallization in separation and/or purifying from the mother liquor, or combinations thereof.It, can be with higher receipts by additional step
Rate obtains psicose, and can reduce the amount for the D-Fructose that can be dropped, to provide economic benefit.
In an exemplary embodiment, contact of the invention can be carried out at pH5.0-9.0,40-90 DEG C, and/or
It carries out 0.5-48 hours.
Specifically, contact of the invention can be carried out in pH6.0 to 8.5, pH6.0 to 8.0 or pH7.0 to 8.0.
In addition, contact of the invention can 40 DEG C -80 DEG C, 40 DEG C -75 DEG C, 40 DEG C -65 DEG C, 50 DEG C -90 DEG C, 50 DEG C -
80℃、50℃-75℃、50℃-65℃、55℃-90℃、55℃-80℃、55℃-75℃、55℃-65℃、60℃-90℃、60
It is carried out at a temperature of DEG C -80 DEG C, 60 DEG C -75 DEG C, 60 DEG C -65 DEG C, 65 DEG C -90 DEG C, 65 DEG C -80 DEG C or 65-75 DEG C.
In addition, contact of the invention can carry out 0.5 hour or longer, 1 hour or longer, 3 hours or longer, 5 hours
Or it is longer or 6 hours or longer, and/or 48 hours or shorter, 36 hours or shorter, 24 hours or shorter, 12 hours or more
It is short, 9 hours or shorter.
Psicose epimerase, metal and carrier described in the method for D-Psicose are prepared in of the invention
It is identical as described in above-mentioned example embodiment.
Another exemplary implementation scheme according to the present invention provides psicose epimerase as described herein, table
The culture of microorganism or the microorganism up to the psicose epimerase turns in psicose preparation for D-Fructose
The purposes of change.
Invention effect
The activity for converting D-Fructose to psicose of psicose epimerase of the invention be it is excellent, have
The high-temperature stability of commercially available degree, and there is quick conversion reaction rate.Therefore, when A Luo ketone of the invention
When sugared epimerase be used to prepare psicose, psicose can be prepared with high efficiency and high yield.
Detailed description of the invention
Fig. 1 is the schematic diagram for expressing the recombinant vector of psicose epimerase (KGDPE), the psicose
Epimerase is made of the amino acid sequence of SEQ ID NO:1 of the invention.
Fig. 2 is about the result for using KGDPE of the invention, being prepared using D-Fructose as the psicose of substrate
HPLC analyzes data.
Fig. 3 A and 3B are to show psicose epimerase according to the figure of the relative activity of temperature, and wherein Fig. 3 A is shown
It is originated from the D-Psicose 3- epimerase of Agrobacterium tumefaciems (Agrobacterium tumefaciens) in the prior art
(ATPE) activity, Fig. 3 B show the activity of KGDPE in the present invention.
Fig. 4 is the figure for showing the relative activity according to pH of KGDPE of the invention.
Fig. 5 is the figure for showing the relative activity of the various metals of addition of KGDPE of the invention.
Embodiment is described in detail
Hereinafter, by embodiment, the present invention will be described in more detail.However, the present invention is not limited to following implementations
Example, and it is to be understood that those skilled in the art can carry out various modifications and change within the scope and spirit of this invention.
Specification of the invention in the whole text in, unless otherwise indicated, for indicating that it is solid that " % " of specific substance concentration refers to
Body/solid (w/w) %, solid/liquid (weight/volume) % and liquid/liquid (volume/volume) %.
Embodiment
The preparation conversion bacterial strain of embodiment 1., the psicose that conversion bacterial strain preparation is originated from the microorganism that Kaistia belongs to are poor
To isomerase.
Selection is expected have psicose epimerase activity (by D-Fructose from Kaistia microorganism belonging to genus be converted to Ah
Lip river ketose) gene, and prepare include the gene recombinant expression carrier and conversion microorganism.
Specifically, to be expected to psicose poor for selection from the gene order of the Genbank Kaistia microorganism belonging to genus registered
To the gene kgd pe of the Kaistia granuli KCTC12575 of isomerase, and the amino acid sequence (SEQ based on the gene
ID NO:1) and nucleotide sequence (SEQ ID NO:2) design and synthesis forward primer (SEQ ID NO:3) and reverse primer
(SEQ ID NO:4).By using the primer of synthesis, use the genomic DNA of Kaistia granuli KCTC12575 as
Template, by carry out PCR reaction (33 circulations: including 94 DEG C 1 minute, 58 DEG C of 30 seconds and 72 DEG C of 1 of 1 minute circulations) amplification
Gene.Using the gene of PCR purification kit (Quiagen) purifying amplification, and it is inserted into using restriction enzyme NdeI and NotI
To construct recombinant vector pET24a (+)-KGDPE (Fig. 1) in pET24a (+) (Novagen, USA).
Recombinant vector is converted by heat-shock transformed (Sambrook and Russell:Molecular Cloning, 2001)
To in e. coli bl21 (DE3), then freezen protective in 50% glycerol and is used.The Strain Designation of conversion is Escherichia coli
BL21 (DE3)/KGDPE is preserved in the international accession center Korean Culture Center according to budapest treaty, preservation day
Phase is on October 20th, 2016, deposit number KCCM11918P.
The preparation and purification of 2. psicose epimerase of embodiment
It, will in order to which e. coli bl21 (the DE3)/KGDPE prepared from embodiment 1 prepares psicose epimerase
E. coli bl21 (DE3)/KGDPE is inoculated into 5ml LB- kanamycins culture medium, and in 37 DEG C, 200rpm shaken cultivation,
Until the absorbance at 600nm reaches 1.5.Then, the culture solution of shaken cultivation is inoculated into 500ml LB- kanamycins culture
In base, when the absorbance at 600nm is 0.7, it is added 0.5mM isopropyl ss-D-1- thiogalactoside (IPTG), and 16
DEG C and 150rpm to cell carry out it is main culture 16 hours.
By main culture solution with 8000rpm centrifugation 20 minutes, cell is only recycled, is washed cell 2 times with 0.85% (w/v) NaCl
Afterwards, with lysis buffer (50mM Tris-HCl, pH7.0~300mM NaCl) lysis, 20 are cracked with sonic wave vibrator at 4 DEG C
Minute.By the liquid through cracking at 4 DEG C, 13,000rpm centrifugations 20 minutes are to recycle supernatant.Then, supernatant is applied to
The Ni-NTA column (Ni-NTA Superflow, Qiagen) balanced in advance with above-mentioned lysis buffer, and continue to make containing 250mM
The buffer (50mM Tris-HCl, 300mM NaCl, pH7.0) of imidazoles is flowed through to obtain the psicose epimerism of purifying
Enzyme (hereinafter referred to as KGDPE).The SDS-PAGE of KGDPE confirms that the size of monomer is about 32kDa.
The active confirmation of embodiment 3.KGDPE
3-1. confirmation is converted to the active of psicose from D-Fructose
In order to confirm whether KGDPE uses D-Fructose to prepare psicose as substrate, by what is prepared in embodiment 2
It is slow that KGDPE (50mM Tris-HCl, pH7.0) is added to the 50mM Tris-HCl containing 50wt%D- fructose and 3mM MnSO4
In fliud flushing (pH8.0), and reacted 6 hours at 55 DEG C.Then, it is reacted in 100 DEG C of heating stoppings in 5 minutes, then passes through HPLC points
The preparation of analysis confirmation psicose.HPLC analysis uses the HPLC for being equipped with Aminex HPX-87C column (BIO-RAD)
(Agilent, USA) refractive index detector (Agilent1260RID) carries out, and wherein mobile phase solvent is water, and temperature is 80 DEG C,
Flow velocity is 0.6ml/min.
As a result, it is thus identified that KGDPE can be used from D-Fructose and prepare psicose (Fig. 2).
3-2. confirms the activity that psicose is converted to from D-Fructose
In order to confirm whether the preparative capacibility of KGDPE is better than the conventional psicose epimerism prepared for psicose
Enzyme (ATPE, SEQ ID NO:5, Korean Patent Publication No. 10-2011-0035805), it is thus identified that from D-Fructose to psicose
Conversion rate.
Specifically, the e. coli bl21 (DE3) converted with recombinant expression carrier pET24a-ATPE is inoculated into contain and blocks that
In the LB culture medium of mycin (concentration is 10 μ g/ml), then enzyme is expressed and purified in the same way as in example 2.
Obtained enzyme is added in the 50mM Tris-HCl buffer (pH8.0) containing 50wt%D- fructose and 3mM MnSO4, In
55 DEG C are reacted 6 hours.Then, it is reacted in 100 DEG C of heating stoppings in 5 minutes, the system of psicose is then analyzed to identify by HPLC
It is standby.HPLC analysis is being carried out under the same conditions with embodiment 3-1.The conversion rate of psicose is calculated as per minute by enzyme system
The amount (mg/min) of standby psicose, and the reaction rate of KGDPE is shown as relative value, wherein the reaction rate value of ATPE
It is set as 100%.
As a result, it is thus identified that compared with when using ATPE, the amount of the psicose prepared per minute when using KGDPE is
117.6%, therefore, when using KGDPE, the conversion rate from D-Fructose to psicose dramatically increases (table 1).
[table 1]
| Enzyme | KGDPE | ATPE |
| Opposite conversion rate (%) | 117.6 | 100 |
The analysis of embodiment 4.KGDPE feature
4-1. is according to temperature analysis enzymatic activity
By KGDPE and D-Fructose substrate at (40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C of condition of different temperatures
With 75 DEG C) under react 2 hours, and compare the enzymatic activity according to temperature.In addition to temperature and other than the reaction time, with embodiment 3-
Identical mode carries out above-mentioned reaction in 1, and measures enzymatic activity with the conversion rate from D-Fructose to psicose.Conversion speed
Rate is calculated as percentage of the weight of the psicose prepared after reaction relative to the weight of substrate (D-Fructose) before reacting.
As a result, KGDPE shows 25% or higher high activity of conversion in all measurement temperature ranges, and confirm
As temperature increases, activity is increased, and observes maximum conversion rate (table 2) under 75 DEG C of maximum temperature.
[table 2]
| Temperature (DEG C) | KGDPE (conversion rate, %) |
| 40 | 26.7 |
| 45 | 27.8 |
| 50 | 28.8 |
| 55 | 29.7 |
| 60 | 30.5 |
| 65 | 31.2 |
| 70 | 32.1 |
| 75 | 32.8 |
The thermal stability analysis of 4-2. enzyme
In order to compare the thermal stability of KGDPE and traditional enzyme ATPE, it is heat-treated at (55 DEG C, 60 DEG C and 65 DEG C) of different temperatures
Each enzyme, and at each heat treatment time (0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours) is to enzymatic
Reason solution is sampled to measure the residual activity of every kind of enzyme.When being reacted by only changing with identical mode in embodiment 3-1
Between, reaction 30 minutes is carried out, and the residual activity of enzyme is measured by the conversion rate from D-Fructose to psicose.
As a result, the half-life period of KGDPE reduces the significant half-life period less than ATPE with temperature is raised, therefore, it was demonstrated that KGDPE
With high thermal stability (Fig. 3 A and 3B).
4-3. analyzes enzymatic activity according to pH
In order to measure enzymatic activity according to pH, react D-Fructose substrate with KGDPE in various pH.At this point, when in addition to reaction
Between and pH value other than, reaction carried out in a manner of identical with embodiment 3-1.
Specifically, enzyme reaction carries out 30 minutes at 55 DEG C, using 50mM potassium phosphate, pH5.0, pH6.0, pH6.5,
PH7.0, pH7.5 and pH8.0, and use 50mM Tris-HCl buffer, pH8.0, pH8.5 and pH9.0.Then, with from D-
The conversion rate of fructose to psicose measures enzymatic activity.
As a result, it is thus identified that compared with maximum activity, KGDPE shows 70% or higher activity in pH6-pH8.5, and
PH8.0 shows highest active (table 3, Fig. 4).
[table 3]
The enzyme activity assay that 4-4. is added according to metal
In order to confirm the activity of the KGDPE according to metal addition, under reaction condition identical with embodiment 3-1, with each
Kind metal salt (LiCl, Na2SO4、MgCl2、NaCl、FeSO4And CaCl2) replace MnSO4, and it is added to final concentration of 3mM.So
Afterwards, enzymatic activity is measured.Control group does not have to metal salt treatment.
As a result, it is thus identified that compared with the control group, Li, Na, Mg, Fe and Ca is added and Mn improves the activity of KGDPE,
In, it can be verified that Mn farthest improves enzymatic activity (table 4 and Fig. 5).
[table 4]
From the above description, it will be appreciated by those skilled in the art that the present invention can realize in other specific forms, without changing
Become its technical idea or essential characteristic.Thus, it should be understood that the embodiment above is all illustrative rather than limit in all respects
Property processed.The scope of the present invention should be interpreted to cover derivative from the meaning and scope of the following claims and their equivalents
All modifications or changes, rather than be described in detail.
Sequence table
<110>CJ Corp.
<120>a kind of novel D-Psicose 3- epimerase and the method for preparing D-Psicose using the enzyme
<130> OPA18334
<150> KR 10-2016-0152947
<151> 2016-11-16
<160> 5
<170> KopatentIn 2.0
<210> 1
<211> 297
<212> PRT
<213> Amino acid sequences of KGDPE
<400> 1
Met Lys Asn Lys Leu Gly Val His Ala Gln Val Trp Val Gly Gly Trp
1 5 10 15
Ser His Ala Glu Ala Glu Arg Ala Ile Ala Ser Thr Ala Ser Leu Gly
20 25 30
Tyr Asp Tyr Ile Glu Ala Pro Ala Leu Asp Pro Ser Leu Ile Asp Ile
35 40 45
Asp Phe Thr Arg Lys Ala Leu Glu Lys His Gly Leu Gly Ile Thr Thr
50 55 60
Ser Leu Gly Leu Asp Asp Ser Cys Asp Ile Ser Ser Gly Asp Pro Asp
65 70 75 80
Lys Lys Ala Arg Gly Gln Ala His Leu Met Lys Val Val Ser Thr Thr
85 90 95
Arg Asp Leu Gly Gly Thr His Ile Thr Gly Ile Leu Tyr Ser Gly Phe
100 105 110
Gln Lys Tyr Phe Thr Pro Ala Thr Pro Glu Gly Val Ala Gly Ala Val
115 120 125
Glu Val Leu His His Val Ala Glu Glu Ala Ala Lys Ser Asn Ile Thr
130 135 140
Leu Gly Leu Glu Val Val Asn Arg Tyr Glu Thr Asn Val Ile Asn Thr
145 150 155 160
Ala Ala Gln Gly Val Glu Leu Cys Lys Arg Val Gly Met Pro Asn Val
165 170 175
Lys Val His Leu Asp Cys Tyr His Met Asn Ile Glu Glu Ala Asp Ala
180 185 190
Glu Arg Ala Ile Ile Asp Thr Gly Asp Tyr Leu Gly Tyr Phe His Thr
195 200 205
Gly Glu Ser His Arg Gly Tyr Leu Gly Thr Gly Ser Ile Asp Phe Thr
210 215 220
Arg Ile Phe Arg Gly Leu Val Lys Ala Asn Tyr Gln Gly Pro Ile Cys
225 230 235 240
Phe Glu Ser Phe Ser Ser Ala Val Ala Gly Glu Pro Leu Ser Gly Ile
245 250 255
Leu Gly Ile Trp Arg Asn Leu Trp Thr Asp Ser Thr Asp Leu Cys Arg
260 265 270
His Ala Met Gln Phe Thr Gln Ala Gln Met Gln Ala Ala Glu Gln Ala
275 280 285
Gln Ser Ile Arg Thr Gly Ala Asp Trp
290 295
<210> 2
<211> 894
<212> DNA
<213> Nucleotide sequences of KGDPE
<400> 2
atgaagaaca agctgggtgt gcacgcacag gtctgggtcg gcggctggag ccatgcggag 60
gcggagcgcg ccatcgccag caccgcctcg ctcggctacg actatatcga ggcgccggcg 120
ctcgacccgt cgctgatcga catcgacttc acccgcaaag cgctggaaaa gcatggtctc 180
ggcatcacga cgtcgctcgg cctcgacgac agctgcgaca tctcctcggg cgatcccgac 240
aagaaggcgc gcggccaggc gcacctgatg aaggtggtct ccaccacccg tgatctcggc 300
ggcacccaca tcaccggtat cctctattcc ggcttccaga aatacttcac gcccgcaacg 360
ccggagggcg tcgccggcgc cgtcgaggta ttgcaccacg tcgccgagga agcggcgaag 420
agcaacatca cgctcggcct cgaggtggtg aaccgctacg agaccaacgt gatcaacacc 480
gccgcccagg gcgtcgagct ctgcaagcgg gtcggcatgc cgaacgtcaa ggtgcacctc 540
gactgctacc acatgaacat cgaggaagcc gacgccgagc gcgccatcat cgataccggc 600
gactatctgg gttatttcca taccggtgaa tcgcatcgcg gctatctcgg caccggctcg 660
atcgacttca cccgcatctt ccgcggcctg gtgaaggcca actaccaggg tccgatctgc 720
ttcgaatcct tctcgtccgc cgtcgccggc gagccgctct ccggcattct cggcatctgg 780
cgcaatctct ggacggattc gaccgatctc tgccgccacg ccatgcagtt cacgcaggca 840
cagatgcagg cggccgagca ggcccagtcg atccgcaccg gcgcggactg gtag 894
<210> 3
<211> 22
<212> DNA
<213> A forward primer of KGDPE
<400> 3
atgaagaaca agctgggtgt gc 22
<210> 4
<211> 20
<212> DNA
<213> A reverse primer of KGDPE
<400> 4
tcaccagtcc gcgccggtgc 20
<210> 5
<211> 289
<212> PRT
<213> Amino acid sequences of ATPE
<400> 5
Met Lys His Gly Ile Tyr Tyr Ser Tyr Trp Glu His Glu Trp Ser Ala
1 5 10 15
Lys Phe Gly Pro Tyr Ile Glu Lys Val Ala Lys Leu Gly Phe Asp Ile
20 25 30
Leu Glu Val Ala Ala His His Ile Asn Glu Tyr Ser Asp Ala Glu Leu
35 40 45
Ala Thr Ile Arg Lys Ser Ala Lys Asp Asn Gly Ile Ile Leu Thr Ala
50 55 60
Gly Ile Gly Pro Ser Lys Thr Lys Asn Leu Ser Ser Glu Asp Ala Ala
65 70 75 80
Val Arg Ala Ala Gly Lys Ala Phe Phe Glu Arg Thr Leu Ser Asn Val
85 90 95
Ala Lys Leu Asp Ile His Thr Ile Gly Gly Ala Leu His Ser Tyr Trp
100 105 110
Pro Ile Asp Tyr Ser Gln Pro Val Asp Lys Ala Gly Asp Tyr Ala Arg
115 120 125
Gly Val Glu Gly Ile Asn Gly Ile Ala Asp Phe Ala Asn Asp Leu Gly
130 135 140
Ile Asn Leu Cys Ile Glu Val Leu Asn Arg Phe Glu Asn His Val Leu
145 150 155 160
Asn Thr Ala Ala Glu Gly Val Ala Phe Val Lys Asp Val Gly Lys Asn
165 170 175
Asn Val Lys Val Met Leu Asp Thr Phe His Met Asn Ile Glu Glu Asp
180 185 190
Ser Phe Gly Asp Ala Ile Arg Thr Ala Gly Pro Leu Leu Gly His Phe
195 200 205
His Thr Gly Glu Cys Asn Arg Arg Val Pro Gly Lys Gly Arg Met Pro
210 215 220
Trp His Glu Ile Gly Leu Ala Leu Arg Asp Ile Asn Tyr Thr Gly Ala
225 230 235 240
Val Ile Met Glu Pro Phe Val Lys Thr Gly Gly Thr Ile Gly Ser Asp
245 250 255
Ile Lys Val Trp Arg Asp Leu Ser Gly Gly Ala Asp Ile Ala Lys Met
260 265 270
Asp Glu Asp Ala Arg Asn Ala Leu Ala Phe Ser Arg Phe Val Leu Gly
275 280 285
Gly
Claims (10)
1. a kind of D-Psicose 3- epimerase is made of the amino acid sequence of SEQ ID NO:1.
2. the D-Psicose 3- epimerase of claim 1, wherein D-Psicose 3- epimerase is by SEQ ID
The polynucleotide sequence coding of NO:2.
3. a kind of polynucleotides for the D-Psicose 3- epimerase for encoding claim 1.
4. a kind of recombinant vector, it includes the polynucleotides of claim 3.
5. a kind of microorganism, wherein having imported the recombinant vector of claim 4.
6. a kind of composition for being used to prepare D-Psicose, it includes: the D-Psicose 3- epimerism of claim 1
Enzyme, the microorganism for expressing D-Psicose 3- epimerase or the microorganism culture.
7. the composition of claim 6, also includes: D-Fructose.
8. a kind of method for preparing D-Psicose comprising: by D-Psicose 3- epimerase, the table of claim 1
The culture of microorganism or the microorganism up to D-Psicose 3- epimerase is contacted with D-Fructose.
9. method for claim 8 wherein the contact is carried out in the pH of 5.0-9.0,40 DEG C -90 DEG C of temperature, or carries out
0.5-48 hours.
10. method for claim 8, further include: the D- for making claim 1 before, after or at the same time is being contacted with D-Fructose
The culture of psicose 3- epimerase, the microorganism for expressing D-Psicose 3- epimerase or the microorganism with
Metal contact.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR20160152947 | 2016-11-16 | ||
| KR10-2016-0152947 | 2016-11-16 | ||
| PCT/KR2017/012970 WO2018093153A1 (en) | 2016-11-16 | 2017-11-15 | Novel d-psicose 3-epimerase and method for preparing d-psicose using same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110462036A true CN110462036A (en) | 2019-11-15 |
| CN110462036B CN110462036B (en) | 2023-07-14 |
Family
ID=62145632
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201780077035.7A Active CN110462036B (en) | 2016-11-16 | 2017-11-15 | Novel D-psicose 3-epimerase and method for preparing D-psicose by using same |
Country Status (10)
| Country | Link |
|---|---|
| US (1) | US11174475B2 (en) |
| EP (1) | EP3543336A4 (en) |
| JP (1) | JP6967599B2 (en) |
| KR (1) | KR101919713B1 (en) |
| CN (1) | CN110462036B (en) |
| AR (1) | AR110093A1 (en) |
| RU (1) | RU2727903C1 (en) |
| TW (1) | TWI666323B (en) |
| UA (1) | UA126473C2 (en) |
| WO (1) | WO2018093153A1 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110904132A (en) * | 2019-11-05 | 2020-03-24 | 吉林中粮生化有限公司 | Coding gene, vector and recombinant cell of D-psicose3-epimerase and application thereof |
| CN111019928A (en) * | 2019-12-11 | 2020-04-17 | 吉林中粮生化有限公司 | Coding gene, vector and recombinant cell of D-psicose 3-epimerase and application thereof |
| WO2021103234A1 (en) * | 2019-11-29 | 2021-06-03 | 浙江工业大学 | D-psicose 3-epimerase mutant, and application thereof |
| CN113302299A (en) * | 2019-12-19 | 2021-08-24 | 大象（株） | Psicose epimerase variants, methods of producing the same, and methods of producing psicose using the same |
| CN114262703A (en) * | 2021-12-31 | 2022-04-01 | 保龄宝生物股份有限公司 | Method for enriching D-psicose 3-epimerase by using membrane and application thereof |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101965509B1 (en) * | 2017-11-15 | 2019-04-03 | 씨제이제일제당 (주) | A Novel D-Psicose 3-Epimerase and Methods for Preparing D-Psicose Using The Same |
| CN116848252A (en) | 2021-12-29 | 2023-10-03 | 大象株式会社 | Novel promoter variants for constitutive expression and uses thereof |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100129865A1 (en) * | 2005-11-15 | 2010-05-27 | Kazuhiko Maruta | Ketose 3-epimerase, its preparation and uses |
| KR20140021974A (en) * | 2012-08-10 | 2014-02-21 | 주식회사 삼양제넥스 | Composition for converting fructose into psicose, and method for producing psicose using the same |
| CN103710330A (en) * | 2014-01-03 | 2014-04-09 | 江南大学 | High-catalytic-activity mutant enzyme for D-allulose 3-epimerase and application thereof |
| CN103849613A (en) * | 2014-01-03 | 2014-06-11 | 江南大学 | Thermal stability improved mutant enzyme of D-psicose 3-epimerase and application thereof |
| CN104160023A (en) * | 2011-08-24 | 2014-11-19 | Cj第一制糖株式会社 | D-psicose 3-epimerase mutant with improved thermal stability, and continuous production of d-psicose using same |
| CN105602879A (en) * | 2016-01-26 | 2016-05-25 | 中国科学院天津工业生物技术研究所 | Genetic engineering strain capable of effectively secreting D-psicose 3-epimerase and construction method and application thereof |
| CN105637089A (en) * | 2013-09-03 | 2016-06-01 | 罗盖特兄弟公司 | Improved variant of d-psicose 3-epimerase and uses thereof |
| CN105802897A (en) * | 2016-05-26 | 2016-07-27 | 江南大学 | D-psicose-3-epimerase producing strain and application thereof |
| CN105849260A (en) * | 2013-12-26 | 2016-08-10 | 株式会社三养社 | Polynucleotide encoding psicose epimerase and method of producing psicose using the same |
| CN109563529A (en) * | 2015-12-23 | 2019-04-02 | Cj第制糖株式会社 | The composition for being used to generate D-Psicose comprising D-Psicose 3- epimerase and salt and the method for generating D-Psicose using the composition |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0822937D0 (en) * | 2008-12-16 | 2009-01-21 | Terranol As | Microorganism |
| KR20110035805A (en) | 2009-09-30 | 2011-04-06 | 씨제이제일제당 (주) | Method of producing d-psicose using immobilized d-psicose 3-epimerase |
| CN108315316B (en) * | 2013-01-08 | 2021-07-20 | 松谷化学工业株式会社 | Ketose 3-epimerase produced by Arthrobacter globiformis |
| KR101455624B1 (en) * | 2013-04-12 | 2014-10-28 | 주식회사한국야쿠르트 | Novel D-psicose-3-epimerase from Clostridium bolteae having production of functional rare sugar D-psicose and production method of D-psicose using thereof |
| KR101455759B1 (en) * | 2013-04-23 | 2014-10-28 | 씨제이제일제당(주) | D-psicose 3-epimerase variants and production method of D-psicose using them |
-
2017
- 2017-11-15 RU RU2019116720A patent/RU2727903C1/en active
- 2017-11-15 CN CN201780077035.7A patent/CN110462036B/en active Active
- 2017-11-15 JP JP2019547057A patent/JP6967599B2/en active Active
- 2017-11-15 EP EP17871550.4A patent/EP3543336A4/en active Pending
- 2017-11-15 US US16/461,286 patent/US11174475B2/en active Active
- 2017-11-15 UA UAA201905494A patent/UA126473C2/en unknown
- 2017-11-15 WO PCT/KR2017/012970 patent/WO2018093153A1/en unknown
- 2017-11-15 KR KR1020170152620A patent/KR101919713B1/en active IP Right Grant
- 2017-11-16 AR ARP170103190A patent/AR110093A1/en unknown
- 2017-11-16 TW TW106139740A patent/TWI666323B/en active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100129865A1 (en) * | 2005-11-15 | 2010-05-27 | Kazuhiko Maruta | Ketose 3-epimerase, its preparation and uses |
| CN104160023A (en) * | 2011-08-24 | 2014-11-19 | Cj第一制糖株式会社 | D-psicose 3-epimerase mutant with improved thermal stability, and continuous production of d-psicose using same |
| KR20140021974A (en) * | 2012-08-10 | 2014-02-21 | 주식회사 삼양제넥스 | Composition for converting fructose into psicose, and method for producing psicose using the same |
| CN104769125A (en) * | 2012-08-10 | 2015-07-08 | 株式会社三养吉尼克斯 | Psicose epimerase and composition for conversion to psicose using same |
| CN105637089A (en) * | 2013-09-03 | 2016-06-01 | 罗盖特兄弟公司 | Improved variant of d-psicose 3-epimerase and uses thereof |
| CN105849260A (en) * | 2013-12-26 | 2016-08-10 | 株式会社三养社 | Polynucleotide encoding psicose epimerase and method of producing psicose using the same |
| CN103710330A (en) * | 2014-01-03 | 2014-04-09 | 江南大学 | High-catalytic-activity mutant enzyme for D-allulose 3-epimerase and application thereof |
| CN103849613A (en) * | 2014-01-03 | 2014-06-11 | 江南大学 | Thermal stability improved mutant enzyme of D-psicose 3-epimerase and application thereof |
| CN109563529A (en) * | 2015-12-23 | 2019-04-02 | Cj第制糖株式会社 | The composition for being used to generate D-Psicose comprising D-Psicose 3- epimerase and salt and the method for generating D-Psicose using the composition |
| CN105602879A (en) * | 2016-01-26 | 2016-05-25 | 中国科学院天津工业生物技术研究所 | Genetic engineering strain capable of effectively secreting D-psicose 3-epimerase and construction method and application thereof |
| CN105802897A (en) * | 2016-05-26 | 2016-07-27 | 江南大学 | D-psicose-3-epimerase producing strain and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| WENLI ZHANG等: "Biochemical characterization of a d-psicose 3-epimerase from Treponema primitia ZAS-1 and its application on enzymatic production of d-psicose", 《J SCI FOOD AGRIC》 * |
| WENLI ZHANG等: "Characterization of a metal-dependent D-psicose 3-epimerase from a novel strain, Desmospora sp. 8437", 《J AGRIC FOOD CHEM》 * |
| 佚名: "登录号：WP_018181373.1", 《GENBANK》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110904132A (en) * | 2019-11-05 | 2020-03-24 | 吉林中粮生化有限公司 | Coding gene, vector and recombinant cell of D-psicose3-epimerase and application thereof |
| WO2021103234A1 (en) * | 2019-11-29 | 2021-06-03 | 浙江工业大学 | D-psicose 3-epimerase mutant, and application thereof |
| CN111019928A (en) * | 2019-12-11 | 2020-04-17 | 吉林中粮生化有限公司 | Coding gene, vector and recombinant cell of D-psicose 3-epimerase and application thereof |
| CN111019928B (en) * | 2019-12-11 | 2022-08-16 | 吉林中粮生化有限公司 | Coding gene, vector and recombinant cell of D-psicose 3-epimerase and application thereof |
| CN113302299A (en) * | 2019-12-19 | 2021-08-24 | 大象（株） | Psicose epimerase variants, methods of producing the same, and methods of producing psicose using the same |
| CN113302299B (en) * | 2019-12-19 | 2023-10-13 | 大象（株） | Psicose epimerase variants, methods of producing the same, and methods of producing psicose using the same |
| CN114262703A (en) * | 2021-12-31 | 2022-04-01 | 保龄宝生物股份有限公司 | Method for enriching D-psicose 3-epimerase by using membrane and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| EP3543336A4 (en) | 2020-06-17 |
| CN110462036B (en) | 2023-07-14 |
| RU2727903C1 (en) | 2020-07-24 |
| AR110093A1 (en) | 2019-02-20 |
| EP3543336A1 (en) | 2019-09-25 |
| WO2018093153A1 (en) | 2018-05-24 |
| UA126473C2 (en) | 2022-10-12 |
| US11174475B2 (en) | 2021-11-16 |
| KR20180055736A (en) | 2018-05-25 |
| JP2019535320A (en) | 2019-12-12 |
| US20200063117A1 (en) | 2020-02-27 |
| KR101919713B1 (en) | 2018-11-19 |
| JP6967599B2 (en) | 2021-11-17 |
| TWI666323B (en) | 2019-07-21 |
| TW201823456A (en) | 2018-07-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110462036A (en) | A kind of novel D-Psicose 3- epimerase and the method for preparing D-Psicose using the enzyme | |
| KR101473918B1 (en) | D-psicose 3-epimerase, manufacturing method thereof and manufacturing method of D-psicose using the same | |
| CN109563499B (en) | Novel thermostable fructose-6-phosphate-3-epimerase and method for producing allose using the same | |
| US11485963B2 (en) | D-Psicose 3-epimerase and method for producing D-Psicose using the same | |
| JP7404537B2 (en) | Allulose epimerase variant, method for producing the same, and method for producing allulose using the same | |
| EP3865574B1 (en) | Allulose epimerase variant, method for producing same, and method for producing allulose using same | |
| CN112746061A (en) | Meso-diaminopimelate dehydrogenase mutants and uses thereof | |
| KR102114865B1 (en) | Novel psicose-6-phosphate phosphatase, composition for producing psicose including the phosphatase, and method for producing psicose using the phosphatase | |
| KR102063908B1 (en) | A novel thermostable fructose-6-phosphate 3-epimerase and methods for producing allulose using the same | |
| KR102254411B1 (en) | Variant of D-allulose 3-epimerase, manufacturing method thereof and manufacturing method of D-alluose using the same | |
| CN105154457B (en) | A kind of sorbitol dehydrogenase gene and its application from pseudomonas syringae | |
| CN114196696A (en) | Recombinase fused with specific short peptide tag and capable of efficiently catalyzing Reb M generation | |
| KR102055875B1 (en) | A novel thermostable fructose-6-phosphate 3-epimerase and methods for producing allulose using the same | |
| KR102682846B1 (en) | Variant of D-allulose 3-epimerase with excellent heat stability, manufacturing method thereof and manufacturing method of D-alluose using the same | |
| CN109735524B (en) | Alkaline pectinase with psychrophilic property and application thereof | |
| CN112867793B (en) | Novel fructose-C4-epimerase and method for preparing tagatose using the same | |
| JP2023554112A (en) | Allulose epimerase mutant with excellent thermostability, method for producing the same, and method for producing allulose using the same | |
| CN118028277A (en) | Aldohexose-2-epimerase and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |